Protein sulfenylation, the redox-based modification of cysteine thiol side chains by hydrogen peroxide (H2O2), is an important mechanism in signal transduction. Dysregulated protein sulfenylation contributes to a range of human pathologies, including cancer. However, efforts to elucidate the diverse roles of protein sulfenylation in physiology and disease have, to date, suffered from a lack of techniques to probe these modifications in native environments. To address this problem, we have recently introduced a new chemical proteomic strategy to detect changes in protein sulfenylation directly in cells. To date, our preliminary studies have identified several novel intracellular protein targets of H2O2 during growth factor signaling, including the epidermal growth factor receptor (EGFR). Specifically, we have discovered that H2O2 directly modifies a cysteine residue within the ATP-binding site of EGFR, and that oxidation stimulates its tyrosine kinase activity, though the biochemical mechanism for this effect remains to be fully elucidated. In this proposal, we will apply our suite of chemical probes and analytical tools to address four major questions of high significance to the fields of redox signaling, chemical biology, and cancer. Aim 1 of the proposal will define the molecular mechanism by which sulfenylation of EGFR regulates its kinase activity. To identify features that dictate selectivity in H2O2-mediated signaling, we will examine sulfenylation, localization, and enzyme activity of EGFR-targeted protein tyrosine phosphatases (PTPs) in Aim 2. We will evaluate additional targets of intracellular H2O2 generated in response to growth factors using target-based and chemical reporter/proteomic methods in Aim 3. While sulfenylation is a reversible modification in cells, the factors that recycle sulfenylated proteinsto their reduced thiol form (RSH) are largely ill defined. We will test candidate reductases responsible for reversible sulfenylation in Aim 4. The development and application of our chemical tools in cells provides an unprecedented opportunity to elucidate mechanisms that govern sulfenylation of proteins. Given that aberrant sulfenylation of proteins has been linked to aggressive cancer phenotypes and that genetic lesions in H2O2-metabolizing enzymes can contribute to tumorigenesis, defining the mechanisms that control reversible protein sulfenylation is vital for understanding human physiology and disease. We anticipate that these studies will define how sulfenylation of proteins regulates signaling networks that underlie cell growth and identify key enzymes that controls desulfenylation. Ultimately, this will facilitate the identification of new biomarkers and therapeutic targets for cancer, as well as produce methodological advances that expand the scope and utility of proteomic technologies for biological and biomedical discoveries.